Transport Activity of the Sodium Bicarbonate Cotransporter NBCe1 Is Enhanced by Different Isoforms of Carbonic Anhydrase

Transport metabolons have been discussed between carbonic anhydrase II (CAII) and several membrane transporters. We have now studied different CA isoforms, expressed in Xenopus oocytes alone and together with the electrogenic sodium bicarbonate cotransporter 1 (NBCe1), to determine their catalytic activity and their ability to enhance NBCe1 transport activity. pH measurements in intact oocytes indicated similar activity of CAI, CAII and CAIII, while in vitro CAIII had no measurable activity and CAI only 30% of the activity of CAII. All three CA isoforms increased transport activity of NBCe1, as measured by the transport current and the rate of intracellular sodium rise in oocytes. Two CAII mutants, altered in their intramolecular proton pathway, CAII-H64A and CAII-Y7F, showed significant catalytic activity and also enhanced NBCe1 transport activity. The effect of CAI, CAII, and CAII mutants on NBCe1 activity could be reversed by blocking CA activity with ethoxyzolamide (EZA, 10 µM), while the effect of the less EZA-sensitive CAIII was not reversed. Our results indicate that different CA isoforms and mutants, even if they show little enzymatic activity in vitro, may display significant catalytic activity in intact cells, and that the ability of CA to enhance NBCe1 transport appears to depend primarily on its catalytic activity

Three-Dimensional Model for the Human Cl−/HCO3− Exchanger, AE1, by Homology to the E. coli ClC Protein

AbstractAE1 mediates electroneutral 1:1 exchange of bicarbonate for chloride across the plasma membrane of erythrocytes and type A cells of the renal collecting duct. No high-resolution structure is available for the AE1 membrane domain, which alone is required for its transport activity. A recent electron microscopy structure of the AE1 membrane domain was proposed to have a similar protein fold to ClC chloride channels. We developed a three-dimensional homology model of the AE1 membrane domain, using the Escherichia coli ClC channel structure as a template. This model agrees well with a long list of biochemically established spatial constraints for AE1. To investigate the AE1 transport mechanism, we created point mutations in regions corresponding to E. coli ClC transport mechanism residues. When expressed in HEK293 cells, several mutants had Cl−/HCO3− exchange rates significantly different from that of wild-type AE1. When further assessed in Xenopus laevis oocytes, there were significant changes in the transport activity of several AE1 point mutants as assessed by changes in pH. None of the mutants, however, added an electrogenic component to AE1 transport activity. This indicates that the AE1 point mutants altered the transport activity of AE1, without changing its electrogenicity and stoichiometry. The homology model successfully identified residues in AE1 that are critical to AE1 transport activity. Thus, we conclude that AE1 has a similar protein fold to ClC chloride channels

Functional assessment of SLC4A11, an integral membrane protein mutated in corneal dystrophies

SLC4A11, a member of the SLC4 family of bicarbonate transporters, is a widely expressed integral membrane protein, abundant in kidney and cornea. Mutations of SLC4A11 cause some cases of the blinding corneal dystrophies, congenital hereditary endothelial dystrophy, and Fuchs endothelial corneal dystrophy. These diseases are marked by fluid accumulation in the corneal stroma, secondary to defective fluid reabsorption by the corneal endothelium. The role of SLC4A11 in these corneal dystrophies is not firmly established, as SLC4A11 function remains unclear. To clarify the normal function(s) of SLC4A11, we characterized the protein following expression in the simple, low-background expression system Xenopus laevis oocytes. Since plant and fungal SLC4A11 orthologs transport borate, we measured cell swelling associated with accumulation of solute borate. The plant water/borate transporter NIP5;1 manifested borate transport, whereas human SLC4A11 did not. SLC4A11 supported osmotically driven water accumulation that was electroneutral and Na⁺ independent. Studies in oocytes and HEK293 cells could not detect Na⁺⁻ coupled HCO3⁻ transport or Cl⁻/HCO3⁻ exchange by SLC4A11. SLC4A11 mediated electroneutral NH3 transport in oocytes. Voltagedependent OH⁻ or H⁺ movement was not measurable in SLC4A11- expressing oocytes, but SLC4A11-expressing HEK293 cells manifested low-level cytosolic acidification at baseline. In mammalian cells, but not oocytes, OH⁻/H⁺ conductance may arise when SLC4A11 activates another protein or itself is activated by another protein. These data argue against a role of human SLC4A11 in bicarbonate or borate transport. This work provides additional support for water and ammonia transport by SLC4A11. When expressed in oocytes, SLC4A11 transported NH3, not NH3/H⁺.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

14-3-3 Proteins and Other Candidates form Protein-Protein Interactions with the Cytosolic C-terminal End of SOS1 Affecting Its Transport Activity

The plasma membrane transporter SOS1 (SALT-OVERLY SENSITIVE1) is vital for plant survival under salt stress. SOS1 activity is tightly regulated, but little is known about the underlying mechanism. SOS1 contains a cytosolic, autoinhibitory C-terminal tail (abbreviated as SOS1 C-term), which is targeted by the protein kinase SOS2 to trigger its transport activity. Here, to identify additional binding proteins that regulate SOS1 activity, we synthesized the SOS1 C-term domain and used it as bait to probe Arabidopsis thaliana cell extracts. Several 14-3-3 proteins, which function in plant salt tolerance, specifically bound to and interacted with the SOS1 C-term. Compared to wild-type plants, when exposed to salt stress, Arabidopsis plants overexpressing SOS1 C-term showed improved salt tolerance, significantly reduced Na+ accumulation in leaves, reduced induction of the salt-responsive gene WRKY25, decreased soluble sugar, starch, and proline levels, less impaired inflorescence formation and increased biomass. It appears that overexpressing SOS1 C-term leads to the sequestration of inhibitory 14-3-3 proteins, allowing SOS1 to be more readily activated and leading to increased salt tolerance. We propose that the SOS1 C-term binds to previously unknown proteins such as 14-3-3 isoforms, thereby regulating salt tolerance. This finding uncovers another regulatory layer of the plant salt tolerance program

Catalytically inactive carbonic anhydrase-related proteins enhance the transport of lactate by MCT1

Carbonic anhydrases (CA) catalyze the reversible hydration of CO2 to protons and bicarbonate and thereby play a fundamental role in the epithelial acid/base transport mechanisms serving fluid secretion and absorption for whole‐body acid/base regulation. The three carbonic anhydrase‐related proteins (CARPs) VIII, X, and XI, however, are catalytically inactive. Previous work has shown that some CA isoforms noncatalytically enhance lactate transport through various monocarboxylate transporters (MCT). Therefore, we examined whether the catalytically inactive CARPs play a role in lactate transport. Here, we report that CARP VIII, X, and XI enhance transport activity of the MCT MCT1 when coexpressed in Xenopus oocytes, as evidenced by the rate of rise in intracellular H+ concentration detected using ion‐sensitive microelectrodes. Based on previous studies, we suggest that CARPs may function as a ‘proton antenna’ for MCT1, to drive proton‐coupled lactate transport across the cell membrane

The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels

The bacterial mechanosensitive channel MscL gates in response to membrane tension as a result of mechanical force transmitted directly to the channel from the lipid bilayer. MscL represents an excellent model system to study the basic biophysical principles of mechanosensory transduction. However, understanding of the essential structural components that transduce bilayer tension into channel gating remains incomplete. Here using multiple experimental and computational approaches, we demonstrate that the amphipathic N-terminal helix of MscL acts as a crucial structural element during tension-induced gating, both stabilizing the closed state and coupling the channel to the membrane. We propose that this may also represent a common principle in the gating cycle of unrelated mechanosensitive ion channels, allowing the coupling of channel conformation to membrane dynamics

The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels

The bacterial mechanosensitive channel MscL gates in response to membrane tension as a result of mechanical force transmitted directly to the channel from the lipid bilayer. MscL represents an excellent model system to study the basic biophysical principles of mechanosensory transduction. However, understanding of the essential structural components that transduce bilayer tension into channel gating remains incomplete. Here using multiple experimental and computational approaches, we demonstrate that the amphipathic N-terminal helix of MscL acts as a crucial structural element during tension-induced gating, both stabilizing the closed state and coupling the channel to the membrane. We propose that this may also represent a common principle in the gating cycle of unrelated mechanosensitive ion channels, allowing the coupling of channel conformation to membrane dynamics

Role of Carbonic Anhydrase IV in the Bicarbonate-Mediated Activation of Murine and Human Sperm

HCO3− is the signal for early activation of sperm motility. In vivo, this occurs when sperm come into contact with the HCO3− containing fluids in the reproductive tract. The activated motility enables sperm to travel the long distance to the ovum. In spermatozoa HCO3− stimulates the atypical sperm adenylyl cyclase (sAC) to promote the cAMP-mediated pathway that increases flagellar beat frequency. Stimulation of sAC may occur when HCO3− enters spermatozoa either directly by anion transport or indirectly via diffusion of CO2 with subsequent hydration by intracellular carbonic anhydrase (CA). We here show that murine sperm possess extracellular CA IV that is transferred to the sperm surface as the sperm pass through the epididymis. Comparison of CA IV expression by qRT PCR analysis confirms that the transfer takes place in the corpus epididymidis. We demonstrate murine and human sperm respond to CO2 with an increase in beat frequency, an effect that can be inhibited by ethoxyzolamide. Comparing CA activity in sperm from wild-type and CA IV−/− mice we found a 32.13% reduction in total CA activity in the latter. The CA IV−/− sperm also have a reduced response to CO2. While the beat frequency of wild-type sperm increases from 2.86±0.12 Hz to 6.87±0.34 Hz after CO2 application, beat frequency of CA IV−/− sperm only increases from 3.06±0.20 Hz to 5.29±0.47 Hz. We show, for the first time, a physiological role of CA IV that supplies sperm with HCO3−, which is necessary for stimulation of sAC and hence early activation of spermatozoa